Method for Identification of Virulence Determinants

ABSTRACT

Disclosed are methods for the determination of virulence determinants in bacteria and in particular bacteria of the genus  Mycobacterium . Also disclosed are compositions and methods for stimulating an immune response in an animal using bacteria and virulence determinants identified by the methods of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/759,287, filed Jan. 11, 2001, which claims the benefit of provisionalapplication 60/175,433 filed Jan. 11, 2000, the disclosures of which arehereby incorporated by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 98-35204-6761 and99-35204-7789 awarded by the United States Department ofAgriculture/CSREES. The government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

Paratuberculosis (Johne's disease) is an incurable, fatal disease ofdomestic and wild ruminants. Mycobacterium paratuberculosis (M.paratuberculosis) is the etiologic agent of this disease. Mycobacteriumavium (M. avium) and M. paratuberculosis are slow-growing faculativeintracellular mycobacteria able to grow in mononuclear phagocytes.DNA-DNA hybridization studies have shown that these micro-organismsbelong to a single genomic species (Hurley et al., Intl. J. Syst.Bacteriol., 38:143-146, 1988), and it has been proposed to reclassify M.paratuberculosis as a subspecies of M. avium (Thorel et al., Intl. J.Syst. Bacteriol. 40:254-260, 1990). Furthermore, all M. paratuberculosisstrains are characterized by the presence of the insertion sequenceIS900 (Green et al., Nucleic Acids Res., 17:9063-9073, 1989), which isabsent from most M. avium strains. Phenotypic differences between M.avium and M. paratuberculosis, such as mycobactin requirement, abilityto grow on egg medium, growth stimulation by pyruvate, and tolerance tocycloserine correlate with variations in pathogenicity and host range(Thorel et al., Intl. J. Syst. Bacteriol. 40:254-260, 1990).

Johne's disease is manifested by chronic diarrhea and weight loss. Aftermonths of diarrhea and wasting, the affected animals either die or areculled. In the United States, the prevalence of M. paratuberculosisinfection in dairy and beef cattle herds has reached 34% in certainareas (Collins et al., J. Am. Vet. Med. Assn. 187:323-329, 1992; Collinset al., J. Am. Vet. Med. Assn. 204:636-641, 1994) and results inmillions of dollars in lost revenues annually. Furthermore, M.paratuberculosis has been tentatively linked to Crohn's disease, achronic granulomatous ileitis in humans. Evidence supporting thepossibility that M. paratuberculosis is the etiologic agent of Crohn'sdisease includes culture of M. paratuberculosis is from intestinaltissue, and amplification by PCR of the subspecies-specific IS900sequence of M. paratuberculosis from biopsy specimens.

Natural infection in cattle is usually acquired in the first months oflife. The age of onset of clinical Johne's disease varies, being mostfrequent during or after the second lactation in dairy cattle. Theprolonged incubation time and the difficulty in diagnosing subclinicalcases facilitate the insidious spread of the infection within a herd.Bacteriologic culture is the most definitive diagnostic method, butrequires substantial time and labor (Stabel, J. Vet. Diag. Invest.,9:375-380, 1997), and it is unable to detect infected animals that donot shed acid-fast bacilli. Progress has been made by combining fecalculture, PCR detection (IS900), and tests for humoral (ELISA) orcellular immunity (IFN-γ test) (Collins, Proceedings of the Fifth Intl.Colloq. Paratuberculosis, Chiodini et al., eds., Intl. Assn. forParatuberculosis, 1997, pp. 232-241.). More recently, a gene unique toM. paratuberculosis (hspX) was identified and has promise as a newdiagnostic tool (Ellingson et al., Mol. Cell Probes 12:133-142, 1998).

Currently, treatment of paratuberculosis in cattle is limited to theextra label use of therapeutic agents (St.-Jean et al., Vet. Clin. N.Am. Food Anim. Pract., 7:793-804, 1991; St.-Jean, Vet. Clin. N. Am. FoodAnim. Pract., 12:417-430, 1996), and no antibiotic treatment isrecommended for clinical cases of Crohn's disease. Even with a prolongeddrug regimen paratuberculosis in cattle is invariably fatal.

Little is known about M. paratuberculosis immunogens and virulencedeterminants (Cocito et al., Clin. Microbiol. Rev. 7:328-345, 1994).Lipoarabinoman (Sugden et al., J. Clin. Microbiol., 29:1659-1664, 1991),glycopeptidolipid 1 (Camphausen et al., Proc. Natl. Acad. Sci. USA,82:3068-3072, 1985), and 35 kDa (p35) antigen (El Zaatari et al., J.Clin. Microbiol., 35:1794-1799, 1997) are three major immunogens.Antigen p35 was recognized by sera from all clinically diseased cattleand by fifteen out of twenty cattle with subclinical diseases. Thisantigen, however, is not specific for paratuberculosis, since it iswidely present in other strains of the M. avium complex. Several proteinantigens have been identified by two-dimensional immunoelectrophoresiswith hyperimmune sera, but only a subset of these antigens arerecognized by sera from animals with paratuberculosis (Gunnarsson andFedstand, Acta Vet. Scand. 20:200-215, 1979). Comparison of thetwo-dimensional gel electrophoretic profiles of M. paratuberculosis andM. avium cells grown in Middlebrook 7H9 medium followed by Western blotanalysis, using antiserum from clinically infected cows, revealed a 42kDa protein which may be specific for M. paratuberculosis (White et al.,Am. J. Vet. Res., 55:1399-1405, 1994). Using the same methodology, AHPCgene products were identified that may be antigenic (Hsieh et al.,Proceed. Fifth Intl. Colloq. Paratuberculosis, Chiodini et al., eds.,1997, pp. 82-87).

M. paratuberculosis antigens that have been cloned include the heatshock proteins HSP65 and HSP70, the transposase form IS900, a putativeserine protease, bacterioferritin, the 34 kDa antigen bearing major Bcell epitopes (reviewed by Stevenson and Sharp, Vet. J. 153:269-286,1997), and more recently, the 35 kDa antigen (El Zaatari et al., J.Clin. Microbiol. 35:1794-1799, 1997) and the fled protein from IS900(Doran et al., Microbiol. 143:547-552, 1997). In addition, a novelextracellular ferric reductase enzyme activity with a potential role inthe evasion of intracellular defense mechanisms has been identified(Homuth et al. Infect. Immun., 66:710-716, 1998). Secreted proteins ofM. paratuberculosis have received attention as potential immune targetsearly in infection. Some of these proteins are present as glyconjugatesand different epitopes in the glycosylated and non-glycosylated moietiesseem to be recognized in cattle and sheep (Mutharia et al, Infect.Immun. 65:387-394, 1997).

Diagnosis and control of paratuberculosis presents a significantchallenge. Although vaccination does reduce clinical signs of Johne'sdisease, it does not prevent losses in milk production (van Schaik etal., Vet. Rec., 139:624-627, 1996). Improved vaccines and diagnostictools are urgently needed. Likewise faster, specific, and more accurateand sensitive diagnostics need to be developed, especially to detectanimals in the early stages of the disease. These tools preferablyshould also be able to discriminate between vaccinated and infectedanimals.

Identification of M. paratuberculosis virulence determinants is acritical step in developing suitable methods of diagnosis and control,and requires a systematic method by which virulence determinants can befound. U.S. Pat. No. 5,783,386 to Jacobs et al. describes a method foridentifying virulence determinants of mycobacterial species involvingthe preparation of a genomic DNA library and constructing shuttlevectors containing inserts from the library constructed. These vectorsare then used to transform a virulent organisms to form recombinants.Virulence determinants are identified by inoculating animals with thetransformed recombinant organisms to select virulent recombinants andthen identifying the DNA sequences that confer virulence. Cavaignac, etal. (Arch. Microbial. 173:229-231, 2000) studied virulence mechanisms inM. paratuberculosis by the introduction of random mutants usingtransposon mutagenesis. Two thousand mutants were screened on the basisof auxotrophy and altered cell wall. Pelicic et al. (U.S. Pat. No.6,096,549) disclose a method for inserting a transposon into amycobacterium strain using a vector containing the sacB gene. Use ofthis vector to identify virulence determinants is also disclosed. Thepresent disclosure teaches an alternative method utilizing transposonmediated mutation and positive selection of mutants by antimicrobialagents that kill growing mycobacteria.

BRIEF SUMMARY OF THE INVENTION

Among the several aspects of the invention is provided a method foridentifying virulence determinants of a bacteria comprising introducingat least one mutation into the genome of a bacteria; culturing themutated bacteria in the presence of an antimicrobial agent that killsgrowing but not non-growing bacteria; selecting surviving bacteria;testing the selected surviving bacteria for virulence; selecting the nonvirulent bacteria; sequencing genetic material from the selected nonvirulent bacteria; determining the site of mutation; and comparing thesequence at the mutated site to the corresponding wild type sequence.

Another aspect provides a composition for immunizing an animal againstbacterial infection comprising a pharmaceutically acceptable carrier,diluent or excipient; and at least one non-virulent strain of bacteriaproduced by the process comprising introducing at least one mutationinto the genome of a bacteria; culturing the mutated bacteria in thepresence of an antimicrobial agent that kills growing but notnon-growing bacteria; selecting surviving bacteria; testing the selectedsurviving bacteria for virulence; and selecting the non-virulentstrains.

Still another aspect provides a composition for immunizing an animalagainst a bacteria comprising a pharmaceutically acceptable carrierdiluent or excipient; and at least one bacterial virulence determinant,the determinant identified by a process comprising; introducing at leastone mutation into the genome of a bacteria; culturing the mutatedbacteria in the presence of an antimicrobial agent that kills growingbut not non-growing bacteria; selecting surviving bacteria; testing theselected surviving bacteria for virulence; selecting the non-virulentstrains; sequencing genetic material from the selected non-virulentbacteria to determine the site of the mutation; and identifying thevirulence determinant based on the site of the mutation.

Yet another aspect provides a method for inducing an immune response inan animal against a bacteria comprising administering to an animal animmune response inducing amount of any of the previously describedcompositions.

Another aspect provides a method for diagnosing infection byMycobacterium paratuberculosis comprising obtaining a sample from ananimal and determining the presence or absence in the sample of abacterial virulence determinant, said determinant identified by themethods described above.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims and accompanying figures where:

FIG. 1 shows Southern blot analysis of M. paratuberculosis K-10 truetransposon mutants.

FIG. 2 shows the location of the BETH-R and BETH-F primers in the Tn5367on and partial results of sequencing. Also shown is the alignment ofnucleotide sequence obtained from mutant GPM207 (SEQ ID NO: 5) usingBETH-F and the xerC gene (GenBank No. Z97369) (SEQ ID NO: 6).

FIG. 3 shows the effects of co-culture with either Bay y 3118 orD-cycloserine on growing and non-growing M. paratuberculosis strain K-10in complete Middlebrook 7H9 medium.

ABBREVIATIONS AND DEFINITIONS

cfu=colony forming unit

pfu=plaque forming unit

MIC=minimal inhibitory concentration

“Albumin dextrose complex” means 2 g glucose, 5 g bovine serum albuminfraction V and 0.85 g NaCl in 100 mL deionized water.

As used herein “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric (2 or more monomers) form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Although nucleotides are usually joined byphosphodiester linkages, the term also includes polymeric nucleotidescontaining neutral amide backbone linkages composed of aminoethylglycine units. This term refers only to the primary structure of themolecule. Thus, this term includes double- and single-stranded DNA andRNA. It also includes known types of modifications for example, labels,methylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.),those containing pendant moieties, such as, for example, proteins(including for e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified fowls of the polynucleotide. Polynucleotidesinclude both sense and antisense strands.

As used herein “polypeptide”, “protein” and “peptide” are usedinterchangeably and refer to a polymer of two or more amino acids.Included within the definition are polypeptides containing one or moreanalogs of an amino acid, including, for example, unnatural amino acids,polypeptides with substituted linkages, as well as modifications knownin the art, both naturally occurring and non-naturally occurring.

As used herein, the term “animal” includes human beings.

As used herein, the term “patient” includes humans and animals.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The following detailed description is provided to aid those skilled inthe art in practicing the present invention. Even so, this detaileddescription should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications, databases and otherreferences cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application, database or other reference were specifically andindividually indicated to be incorporated by reference.

The present invention relates to the method for identification ofvirulence determinants in bacteria particularly in members of the genusMycobacteria. The genus Mycobacteria includes the species M. phlei, M.smegmatis, M. africanum, M. fortuitum, M. marinum, M. ulcerans, M.tuberculosis, M. bovis, M. microti, M. avium, M. paratuberculosis, M.leprae, M. lepraemurium, M. intracellulare, M. scrofulaceum, M. xenopi,M. genavense, M. kansasii, M. simiae, M. szulgai, M. Haemophilum, M.asiaticum, M. amlmoense, and M. shimoidei. In one embodiment, themycobacteria are slow growing mycobacteria. Any virulent strain of aspecies of mycobacterium that is slow growing and capable of beingmaintained in vitro can be used in the practice of the presentinvention. Examples of slow growing mycobacteria include M.tuberculosis, M. bovis, M. africanum, M. marinum, M. avium, and M.paratuberculosis. In one embodiment, strains of M. paratuberculosis areused and in particular the virulent strain K-10 of M. paratuberculosis.

Once a particular strain or strains has been chosen, mutations areintroduced into the genome of the selected bacteria. Numerous means forinserting mutations into the genome of bacteria are known in the art andcan be found in standard references such as Sambrook et al. MolecularCloning, 2nd ed., Cold Spring Harbor Press, 1989 and Ausubel et al.,Short Protocols in Molecular Biology, 3rd ed., Wiley, 1995. A discussionof methods of mutagenesis directed particularly to mycobacteria can befound in Jacobs et al., Methods in Enzymology, 204:537-555, 1991 andPelicic et al., Molec. Microbiol., 28:413-420, 1998. Mutationsintroduced can be site directed to a particular gene or sequence ofinterest, or the location of the mutations can be random. If randommutation is used, it is preferred that the mutations be approximatelyevenly distributed throughout the genome. Two particularly useful formsof mutagenesis are allelic exchange mutagenesis and transposonmutagenesis although other forms of mutagenesis such as chemical andenzymatic (e.g. restriction enzyme mediated integration) mutagenesis canbe used.

In allelic exchange mutagenesis, the gene or sequence of interest isdisrupted using homologous recombination such that the functional alleleis replaced with an inactivated copy. Following introduction of theinactivated copy, the virulence of the transformed bacteria can becompared to the parental strain. The efficiency of allelic exchangemutation is improved through the use of counter selection strategieswhich eliminate transformants retaining the delivery vector. Successfulallelic exchange mutations have been achieved in mycobacteria (U.S. Pat.No. 6,096,549; Balasubramanian et al., J Bacterol. 178:273-279, 1996;McFadden. Molec. Microbiol., 21:205-211, 1996; Pelicic et al., Proc.Natl. Acad. Sci. USA, 94:10955-10960, 1997).

In one embodiment, the introduction of mutations is by transposonmutagenesis. Transposon mutagenesis occurs by the insertion of a mobileelement called a transposon into the gene or sequence of interest, thusdisrupting its function. An advantage of transposon mutagenesis is thatno previous assumptions need to be made regarding the identity of thegene or sequence to be disrupted. In general, a transposon contains aninverted repeat sequence at the 5′ and 3′ ends and a gene or genesencoding transposase enzyme(s) between the inverted repeats. Intransposon mutagenesis, the transposon is removed from a vector andinserted or transposed into the chromosome of a bacteria to be mutated.As used herein, the term “transposon” is a general term and encompassesboth non-mutated and mutated transposons. Thus, the term includestransposons in which a portion of the nucleotide sequence has beendeleted and/or replaced, and/or wherein the transposon containsadditional DNA sequences. Transposon mutagenesis has been successfullyused in mycobacteria (Martin et al., Nature, 345:739-743, 1990; Guilhotet al., J Bacteriol. 176:535-539, 1994; Pelicic et al., Proc. Natl.Acad. Sci USA, 94:10955-10960, 1997; Bardarov et al., Proc. Natl. Acad.Sci. USA, 94:10961-10966, 1997). In one preferred embodiment, transposonmutagenesis is accomplished by use of the transposable element Tn5367, asingle-unit transposon which carries a kanamycin-resistance marker andthe M. smegmatis insertion sequence IS 1096 (Cirillo et al., J.Bacterol. 173:7772-7780, 1991; McAdam et al., Infect. Immunol.,1004-1012, 1995).

Introduction of vectors useful in the practice of the present inventioncan be accomplished by any suitable method known in the art. Variousmethods are known for the introduction of DNA into bacterial cells andinclude, for example, calcium phosphate transfection, DEAE-dextranmediated transfection, Polybrene, protoplast fusion, liposomes, phageinfection, conjugation, and electroporation. Commonly, introduction ofvectors for allelic exchange or transposon mutagenesis in mycobacteriais by either electroporation or phage infection. In one preferredembodiment, introduction of the DNA is by phage infection and inparticular by the use of the TM4 thermosensitive transposon deliveryshuttle plasmid phAE94 (Bardarov et al. Proc. Natl. Acad. Sci. USA,94:10961-10966, 1997).

In general, a shuttle plasmid comprises a bacteriophage DNA into which aplasmid sequence has been inserted into a nonessential region. In oneembodiment, the bacteriophage is from a mycobacteriophage and theplasmid sequence is a cosmid sequence, preferably an E. coli cosmidsequence. Shuttle plasmids can, therefore, replicate in bacteria as aplasmid/cosmid or as a phage. The inserted plasmid/cosmid DNA ispreferably flanked by restriction sites not found in the bacteriophageDNA, so that the inserted DNA can be easily excised. In one embodiment,a cosmid containing a transposon is obtained and inserted into thebacteriophage backbone using standard methods of cosmid cloning(Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Press,1989).

The sequence containing the transposon can, and usually does, alsocontain a selection marker sequence. Typically, this sequence encodes aprotein necessary for the survival or growth of the host celltransformed with the shuttle plasmid. Examples of suitable markers forprokaryotic cells include tetracycline, kanamycin, and ampicillinresistance.

In one embodiment, the shuttle plasmid is a temperature sensitiveplasmid. Temperature sensitive shuttle plasmids are those whichreplicate and form plaques at a permissive temperature, but do notundergo a lytic cycle and so do not form plaques at a non-permissivetemperature.

Once the temperature sensitive shuttle plasmid has been produced it canbe introduced into a bacterial host which will allow growth of theshuttle plasmid as a lytic bacteriophage at a permissive temperature.Introduction of the shuttle plasmid into the host cell can be by anymethod suitable for the introduction of DNA into a bacterial hostincluding those discussed above. Introduction of the shuttle plasmid andculture at a permissive temperature results in the production of largenumbers of bacteriophage particles. The bacteriophages are isolatedusing standard techniques and then used to infect susceptible bacteriaat a non permissive temperature. At the non-permissive temperature,rather than causing lysis of the infected bacteria, the shuttle plasmidgives rise to bacterial transductants which can then be selected on thebasis of a selection marker, if present.

In one preferred embodiment, introduction of the DNA is by the use ofthe TM4 thermosensitive transposon delivery shuttle plasmid phAE94(Bardarov et al. Proc. Natl. Acad. Sci. USA, 94:10961-10966, 1997). Thisshuttle plasmid contains the transposon Tn5367 which is a derivative ofthe insertion sequence IS1096 from M. smegmatis and carries the aph geneconferring kanamycin resistance. In this embodiment, phAE94 ispropagated in M. smegmatis at a permissive temperature and the resultingmycobacteriophage used to infect susceptible mycobacteria at anon-permissive temperature. Mycobacteria undergoing transformation arethen selected by kanamycin resistance.

Once one or more mutations have been introduced into the genome, aselection method is employed in order to select those bacteria which themutation has disrupted a gene or nucleic acid sequence potentiallyinvolved in virulence. Various methods can be employed to determinevirulence. For example, suitable host animals can be inoculated with themutated bacteria and monitored for development of disease symptomsand/or replication of the organisms injected. Alternatively, selectioncan be based on the use of a chemical, and in particular, anantimicrobial, the effect of which is associated with a trait related tovirulence. Since bacterial growth is a necessary part of virulence, inone embodiment the selection is based on the ability of an antimicrobialto kill growing mycobacteria while having a reduced or no effect onnon-growing bacteria. Thus, antimicrobials that interfere with processesinvolved in bacterial growth and in particular DNA replication arepreferred. Although not necessary to practice the present invention, itis preferred that the antimicrobial agent used be able to entereukaryotic cells, thus allowing screening of transformed bacteria inintracellular culture systems, e.g. macrophage culture. In oneembodiment, the antimicrobial is a quinolone. In another embodiment, theantimicrobial is a fluoroquinolone. In yet another embodiment, theantimicrobial is Bay y 3118. Bay y 3118 has been reported to killgrowing but not non-growing Mycobacterium avium within human macrophages(Bermudez et al., FEMS Microbial. Lett. 1787:19-26, 1999). In stillanother embodiment, the antimicrobial is D-cycloserine (Cáceres et al.,J. Bacteriol. 179:5046-5055, 1997).

The exact amount of the antimicrobial used will vary with the particularsubstance and organism in question. In general, the concentration of theantimicrobial should exceed the approximate minimal inhibitoryconcentration (MIC) for the particular organism being studied.Determination of MIC is routine in the art and can be accomplished bythe skilled technician without undue experimentation. In one embodiment,in which M. paratuberculosis strain K-10 and Bay y 3118 are used, theMIC is approximately 0.015 μg/mL. In another embodiment, theconcentration of Bay y 3118 is 5× the MIC. In still another embodimentin which M. paratuberculosis strain K-10 and D-cycloserine are used theMIC is approximately 25 μg/mL. In yet another embodiment, theconcentration of D-cycloserine is 5× the MIC.

In general, screening is accomplished by culturing the mutated bacteriain the presence of the antimicrobial agent. As will be apparent to thoseskilled in the art, the exact culture conditions will vary with theorganism being studied, When M. paratuberculosis is used, either brothor macrophage culture can be used. Methods for the culture ofmycobacteria are well known in the art and can be found, for example, inJacobs et al., Methods in Enzymology, 204:537-555, 1991; Foley-Thomas etal. Microbiology 141:1173-1181, 1995; and Williams et al., J. Clin.Microbiol. 37:304-309, 1999. In one embodiment, mycobacteria are grownin Middlebrook 7H9 broth. As is known in the art and described in thereferences cited above, Middlebrook 7H9 broth is supplemented dependingon the particular species of mycobacteria cultured. Alternatively, anintracellular culture system such as a macrophage or amoeba (Cirillo etal., Infect. Immunol. 65:3759-3767, 1997) culture system can be used.Mycobacteria naturally infect macrophages as part of the diseaseprocess. Thus, mycobacteria can be cultured in macrophages. In thissystem, mycobacteria, preferably in a single cell suspension, are addedto a culture of macrophages from a suitable species. The macrophagesused can be recently collected from blood using known, standard methodsor can be from a macrophage cell line. The mycobacteria are allowed toinfect the macrophages. The infected macrophages are then maintainedunder suitable culture conditions so that the mycobacteria survive andgrow within the macrophages.

The antimicrobial selection agent is added to the culture system at aconcentration that exceeds the MIC and the culture continued for aperiod of time sufficient to kill the multiplying bacteria. It will beapparent to those skilled in the art that the exact amount of timerequired will vary with such well known factors as the amount and typeof selection agent used, the culture system, and the species ofmycobacteria. In general, the selection conditions can be optimized byculturing bacteria for various times and at various concentrations ofantimicrobials to determine the combination of times and concentrationswhich effectively kill growing, but not non-growing bacteria. Suchoptimization can readily be achieved by the skilled technician withoutundue experimentation. After the selection period, the survivingbacteria are collected, individual bacteria isolated, by for exampleclonal streaking, and the clones expanded using standard culturemethods. If desired following expansion, the selected bacteria can befurther characterized by genetic, biochemical, and animal testing.

Characterization can include determination of whether the transposon hasbeen incorporated into the genome. This can be accomplished using wellknown techniques such as Southern blotting, dot or slot blots, and insitu hybridization to bacterial chromosomes, using a polynucleotideprobe complimentary to the transposon used. Information regarding thelocation of the transposition and the gene or sequence disrupted can beobtained by sequencing. In general, this can be accomplished byobtaining purified genomic DNA from transfected bacteria and cutting theDNA with restriction enzymes that do not cut within the transposon. Therestriction fragments obtained can be cloned into a cloning vector usingstandard techniques and amplified for sequencing. Any known method ofsequencing can be used. In one embodiment, sequencing is accomplished bycycle sequencing outward from the transposon. Once the sequenceinformation has been obtained, the nucleic acid sequences or deducedamino acid sequences can be compared to sequences in publicly availabledatabases such as those maintained by the National Center forBiotechnology Information at http://www.ncbi.nlm.nih.gov/, the EuropeanBioinformatics Institute at http://www.ebi.ac.uk/, The Institute forGenomic Research at http://www.tigr.org, The Sanger Centre athttp://www.sanger.ac.uk/Projects/, The Computational Biology Center ofthe University of Minnesota Microbial Genome Project athttp://cbc.umn.edu/, and the Institute Pasteur athttp://genolist.pasteur.fr/. Based on sequence homology, the identity ofthe gene or sequence disrupted by the insertion can be determined andthus the virulence determinant identified.

Alternatively or additionally information can be obtained by biochemicalstudies, especially by auxotrophic analysis. Auxotrophic mutants aremutants that require a nutrient or substance not required by the parentorganism from which the mutant was derived. Determination of auxotrophicmutants can be made by comparing growth on complete and incompletegrowth medium. For those mutants showing no or reduced growth onincomplete medium, the missing nutrients can be individually added backuntil growth comparable to that seen with complete medium is obtained.Methods for selecting auxotrophic mutants are well known in the art andcan be accomplished by the skilled technician without undueexperimentation.

Whether the selected mutants lack virulence can be tested by inoculatingsusceptible animals with the selected organism and determining if theorganism results in clinical symptoms or if the organism multiples andspreads beyond the site of inoculation. The animal inoculated can be thenatural host for the organism or it can be a animal model. In oneembodiment, the mouse model and in particular the beige mouse (Whippleet al. Proc. 3rd Intl. Colloq Paratuberculosis, Intl. Assn.Paratuberculosis, pp. 551-552) is used for virulence testing. Ingeneral, the animal is inoculated with a quantity of the organismsufficient to result in infection. Inoculation can be oral, parenteralor any other suitable method. The exact amount of the organisminoculated will vary with well known factors such as the species, sizeand age-of the animal. Determination of the proper quantity of organismto be administered can be determined by one of ordinary skill in the artwithout undue experimentation. At various times after inoculation, theanimals are sacrificed and various organs examined for the presence ofthe organism. If the organism is not detected, it can be assumed thatthe gene or sequence disrupted is a virulence determinant.

Confirmation that the sequence identified is a virulence determinant canbe obtained by a complementation study. In a complementation study, thewild type sequence is reintroduced into the mutated organism, forexample by introduction of an expression vector, and the effect onvirulence determined. If the disrupted sequence is associated withvirulence, replacement of the sequence should restore virulence. Ingeneral, the disrupted gene or sequence is inserted into a suitableexpression vector using standard techniques in molecular biology foundin standard references and described herein. The expression vector isthen introduced into the mutant organism and the virulence of thecomplemented organism is tested as described above.

The method of the present invention can be used to create strains ofmycobacteria which are non-virulent or have reduced virulence. As usedherein the term attenuated refers to mutated strains whose virulence isreduced as compared to the same organism in its non-mutated form. Suchattenuated mycobacteria can be used in compositions for the treatmentand/or prophylaxis of diseases caused by or associated with theorganism. This includes compositions designed to stimulate an immuneresponse against a organism, for example, a vaccine. Attenuated strainsproduced by the method of the present invention are thought to beparticularly useful because they make it possible to differentiatebetween individuals who test positive for the presence of mycobacteriadue to vaccination or natural infection based on the presence of theintroduced transposon.

Likewise, polypeptides encoded by sequences identified by the method ofthe present invention as being involved in virulence can be used tostimulate an immune response in an animal. In this embodiment, thesequence identified as described herein is placed into an expressionvector. If desired, the polypeptide sequence identified can be fused orassembled with additional amino acids to form a fusion protein. Suchfusion proteins can be used to increase the solubility or antigenicityof the virulence polypeptide. Methods for the production of fusionproteins are well known in the art and can be found in standardmolecular biology references. The expression vector is, in turned,introduced into a suitable prokaryotic or eukaryotic host cell and theencoded polypeptide expressed. The resulting polypeptide is thenpurified using standard biochemical techniques from lysates of the hostcells or from culture medium containing the host cells.

The compositions can be administered to any animal which can becomeinfected with a species of mycobacteria. In one embodiment, the animalis a ruminant animal, more preferably a Bovidae and more preferablystill a member of the genus Bos. When the composition comprises wholemycobacteria, the bacteria can be live or they can be killed by anysuitable means such as heating, chemical treatment, or disruption of thebacterial cell. Bacteria can be in a preserved state such as in alyophilized form which may or may not be reconstituted prior toadministration. The compositions can comprise attenuated bacteria alone,virulence polypeptides alone, or a combination of attenuated bacteriaand virulence polypeptides.

The compositions of the present invention can be administered by avariety of routes and methods. Suitable routes and methods ofadministration include orally, parenterally, by inhalation spray,rectally intradermally, transdermally, or topically in dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. The termparenteral as used herein includes subcutaneous, intravenous,intramuscular, or intrasternal injection, or infusion techniques. In oneembodiment, the complexes are administered by injection. In anotherembodiment, the compositions are administered orally. In yet anotherembodiment, the compositions are administered by inhalation. Methods forthe formulation of drugs is well known in the art and is discussed in,for example, Hoover, John E., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L.Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid are usefulin the preparation of injectables. Dimethyl acetamide, surfactantsincluding ionic and non-ionic detergents, and polyethylene glycols canbe used. Mixtures of solvents and wetting agents such as those discussedabove are also useful.

Suppositories for rectal administration of the compositions discussedherein can be prepared by mixing the active agent with a suitablenon-irritating excipient such as cocoa butter, synthetic mono-, di-, ortriglycerides, fatty acids, or polyethylene glycols which are solid atordinary temperatures, but liquid at the rectal temperature, and whichwill therefore melt in the rectum and release the composition.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, thecompounds of this invention are ordinarily combined with one or moreadjuvants appropriate to the indicated route of administration. Ifadministered per os, the compounds can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents such as sodium citrate, or magnesiumor calcium carbonate or bicarbonate. Tablets and pills can additionallybe prepared with enteric coatings.

For therapeutic purposes, formulations for parenteral administration canbe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. These solutions and suspensions can beprepared from sterile powders or granules having one or more of thecarriers or diluents mentioned for use in the formulations for oraladministration. The compounds can be dissolved in water, polyethyleneglycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil,sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.Other adjuvants and modes of administration are well and widely known inthe pharmaceutical art.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

The amount of attenuated organism and/or virulence determinants that canbe combined with the carrier materials to produce a single dosage formwill vary depending upon the patient or animal and the particular modeof administration. Compositions of the present invention can be given asa single administration or in multiple administrations over a period oftime. If desired, after the initial administration or series ofadministrations, additional periodic administrations (e.g. boosters) ofthe composition can be given.

The attenuated organisms and/or virulence determinants can beadministered in combination with a pharmaceutically acceptable immunesystem stimulant or adjuvant. Examples of such immune system stimulantsor adjuvants include, but are not limited to, Alum (aluminum phosphateor aluminum hydroxide), Freund's adjuvant, calcium phosphate, berylliumhydroxide, dimethyl dioctadecyl ammonium bromide, saponins, polyanions,e.g. poly A:U, Quil A, inulin, lipopolysaccharide endotoxins, liposomes,lysolecithins, zymosan, propionibacteria, mycobacteria, and cytokines,such as, interleukin-1, interleukin-2, interleukin-4, interleukin-6,interleukin-12, interferon-α, interferon-γ, granulocyte-colonystimulating factor.

In another embodiment, the virulence determinants identified by themethod of the present invention are used for diagnosis of mycobacterialinfection. Biological samples can be obtained from a subject suspectedof suffering from a mycobacterial infection. Biological samples includeany sample of tissue or fluid isolated from an individual. Examples ofbiological samples, include, but are not limited to plasma, serum,spinal fluid, lymph fluid, sections of skin, respiratory, intestinal,and genitourinary tracts, tears, saliva, mucus, milk, blood cells,tumors, tumor cells, and organs. Biological samples also include samplesobtained from in vitro cell culture, for example, cells grown inculture, including putatively infected cells and recombinant cells, cellcomponents, and conditioned media resulting from the culture of cells inculture medium.

In one embodiment, diagnosis is made based on the presence ofpolynucleotide sequences identified by the method of the presentinvention. In this embodiment, a biological sample which containsnucleic acids is obtained from an individual. Polynucleotides whether inthe form of DNA or RNA are obtained from the sample using wellestablished techniques which are known to those in the art and can befound in standard molecular biology references; such as those citedherein. In one embodiment, diagnosis is made based on hybridization ofspecific polynucleotide probes to sequences encoding virulencedeterminants identified by the present invention. Probes should be ofsufficient length to provide specificity. In general, probes are atleast 4 nucleotides in length, more preferably at least 8 nucleotides inlength, even more preferably at least 12 nucleotides in length, and morepreferably still at least 20 nucleotides in length. Probes used can beobtained from mycobacteria using standard methods, for example, by theuse of restriction enzyme digestion to obtain suitable nucleic acidfragments which are then inserted into a cloning vector, which is inturn introduced into a suitable host cell. The host cells are then grownunder conditions allowing replication of the cloning vector, and thedesired sequences isolated and used as probes. Alternatively, probes canbe produced by chemical synthesis in automated systems by any suitablemethod, for example, the phosphoramidite method of Beaucage andCarruthers (Teta. Letts., 22:1859-1862, 198). When used forhybridization, it is desirable that the probes be completelycomplementary to the sequence to be detected, but probes which exhibitonly partial complementarity can be used. Hybridization probes can, andoften do, contain detection moieties. Such detection moieties include,but are not limited to radioactive labels, such as radionuclides,fluorophores or fluorochromes, peptides, enzymes, vitamins and steroids.

In order to insure specificity hybridizations should be conducted underhighly stringent conditions. As is recognized in the art stringency is acombination of many factors such as temperature and the composition ofthe hybridization and wash solutions. Thus, many different conditionscan result in the same degree of stringency. In general, highlystringent conditions are achieved by hybridization in a solution of6×SSC or SSPE at a temperature 20-25° C. below the melting temperature(T_(m)) for DNA-DNA hybrids and 10-15° C. below the T_(m) for DNA-RNAhybrids followed by washing in 0.1×SSC or SSPE at 42° C. Even higherstringency conditions can be achieved by washing in 0.1×SSC or SSPE at50-65° C.

In situations where the amount of nucleic acid which can be obtainedfrom the sample is small, it may be desirable to amplify the sequencesof interest by methods such as the polymerase chain reaction (PCR), theligase chain reaction (LCR) (see, Wu and Wallace, Genomics, 4:560-569,1989; Landegren et al., Science, 241:1077-1080, 1988), transcriptionamplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173-1177,1989), self-sustained sequence replication (Guatelli et al., Proc. Natl.Acad. Sci. USA, 87:1874-1878, 1990), and nucleic acid based sequenceamplification (NASBA). In one preferred embodiment, amplification is byPCR. Optimization of conditions for conducting PCR must be determinedfor each reaction and can be accomplished without undue experimentationby one of ordinary skill in the art. In general, methods for conductingPCR can be found in U.S. Pat. Nos. 4,965,188, 4,800,159, 4,683.202, and4,683,195; Ausbel et al., eds., Short Protocols in Molecular Biology,3^(rd) ed., Wiley, 1995; and Innis et al., eds. PCR Protocols, AcademicPress, 1990.

Diagnosis of infection by the presence of sequences identified by themethod of the present invention can be achieved by visualization of PCRproducts of a characteristic size. Individuals testing positive due tonatural infection can be differentiated from individuals who havereceived attenuated vaccine by the presence of the transposon which willresult in an increase in the size of the amplification product.Alternatively, the amplification products could be detected byhybridization to specific probes as described above.

In another embodiment, diagnosis is made by detection of polypeptidesencoded by the sequences identified by the method of the presentinvention. In one embodiment, such detection is by an immunologicalassay. Various immunological assays known in the art can be used,including but not limited to competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in vivoimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, immunoprecipitation reactions, agglutinationassays (e.g., gel agglutination assays, hemagglutination assays),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc. In one embodiment, antibodybinding is detected by detecting a label on the primary antibody. Inanother embodiment, the primary antibody is detected by detectingbinding of a secondary antibody or reagent to the primary antibody. In afurther embodiment, the secondary antibody is labeled.

Antibodies, either polyclonal or monoclonal, against polypeptidesidentified by the method of the present invention for use inimmunological assays can be produced by known methods. If polyclonalantibodies are desired, an animal is immunized with a virulencepolypeptide identified by the method of the present invention. Thecomposition used for immunization can comprise the polypeptide alone ormay further comprise an adjuvant or other immune system stimulant.Further, the polypeptide can be conjugated to a carrier molecule or bepart of a fusion protein in order to increase antigenicity. Followingimmunization, serum is collected from the immunized animals and treatedaccording to well established methods. If desired, the antibody can bepurified using known techniques such as affinity chromatography. Methodsfor the production of polyclonal antibodies are well known in the artand can be found for example in Ausubel et al., Short Protocols inMolecular Biology, 3rd ed. Wiley, 1995.

Likewise, monoclonal antibodies can be produced using well knowntechniques. In general, immortal antibody producing cell lines arecreated by cell fusion, direct transformation of B lymphocytes withoncogenic DNA, or transfection of Epstein-Barr virus. Panels ofhybridomas can then be screened for the production of suitablemonoclonal antibodies using standard techniques. Methods for theproduction of monoclonal antibodies are well known in the art and can befound for example in U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783;4,444,887; 4,466,917; 4,472,500; 4,491,632; and 4,493,890.

Also encompassed by the present invention are diagnostic kits. Such kitscan contain probes, primers and/or antibodies for the detection ofpolynucleotides or polypeptides identified by the method of the presentinvention. The probes, primers and antibodies may, optionally,incorporate a detection moiety such as those described herein. The kitsmay further comprise buffers, reagents and instructions necessary forthe use of the contents of the kits to detect the presence of thepolynucleotides and/or polypeptides.

EXAMPLES Example 1 Bacterial Strains, Growth Conditions, and RecombinantDNA

A virulent clinical isolate of M. paratuberculosis (strain K-10) wasused. M. paratuberculosis cultures were grown standing at 37° C. in acomplete growth medium comprising Middlebrook 7H9 broth enriched witholeic acid albumin dextrose complex, 0.4% casamino acids containingvitamins 40 μg/mL L-tryptophan, 0.5 μg/mL mycobactin J (Allied Monitor,Fayette, Mo.), and 0.05% Tween 80. Escherichia coli DH5α cells, used ascloning hosts were grown on Luria-Bertani (LB) agar or brothsupplemented with 50 μg/mL kanamycin. The conditionally replicating(temperature sensitive) recombinant mycobacteriophage phAE94 (Bardarovet al., Proc. Natl. Acad. Sci. USA, 94:10961-10966, 1997) used todeliver the transposon Tn5367 (McAdam et al., Infect. Immun.63:1004-1012, 1995) was propagated in Mycobacterium smegmatis mc²155 at30° C. as has been described previously. (Bardarov et al. Proc. Nat.Acad. Sci. USA, 94:10961-10966, 1997). The transposon Tn5367 is aderivative of the insertion sequence IS1096 from M. smegmatis andcarries the aph gene conferring kanamycin resistance.

Example 2 Transposon Mutagenesis

M. paratuberculosis cultures were grown to approximately 1.5×10⁸ cfu/mL(OD₆₀₀ 0.38-0.75). Cultures (50 mL) were concentrated by centrifugationand resuspension in 1 mL of MP buffer (50 mM Tris-HCL, pH 7.6, 150 mLNaCl, 2 mM CaCl₂). Bacterial cells and phage were incubated at anon-permissive temperature (37° C.) at the ratios and for the timesindicted in Table 1. After completion of the adsorption time, 2 mL ofprewarmed stop buffer (MP buffer containing 20 mM sodium citrate and0.2% Tween 20) was added to prevent further phage infections. Kanamycinresistant (kan^(r) olonies were selected on complete growth mediumwithout Tween plus 15 g/L agar containing 50 μg/mL kanamycin at 37° C.In five independent experiments, 5,620 transductants were obtained(Table 1). In further experiments, the number of transductants wasincreased to 13,526. This provides a representation of approximately 95%of the genome. Probability (%) values for the representation of themutant pool were calculated using the formula ln(1−P)=N×ln(1−1/4500)assuming a genome size of 5.0 Mb (allowing a 5% underestimation errorfor the reported size) and 4500 target genes and where N=13479 forP=95%.

To verify that transposition events had occurred in these mutants,genomic DNA was isolated using standard techniques (Sambrook et al.,Molecular Cloning, 2nd ed., Cold Spring Harbor Press, 1989) and digestedwith PstI which does cleave the transposon. The digests were thenhybridized overnight under stringent conditions of 65° C. in Rapid-hybbuffer (Amersham-Pharmacia, Piscataway, N.J.) using radiolabeled plasmidpYUB285 which carries the transposon as a probe followed by two, 20minute washes in 2×SSC at 28° C., a 15 minutes wash in 0.5×SSC at 65° C.and a final 15 minute wash in 0.1×SSC at 65° C. Each mutant gave, asexpected, one hybridizable band with at least four distinct sizesobserved (FIG. 1). This confirmed that transpositions had occurred andthat each transformant represented transpositions at differentchromosomal locations. Furthermore all mutants gave the same pattern ofIS900 hybridizable bands as expected for derivatives of strain K-10, andnone of the samples hybridized with TM4 DNA indicating thathybridization was not due to the residual presence of vector phage DNA.

Example 3 Subcloning and DNA Sequencing

Chromosomal DNA from transposon mutant GPM207 and pACYC184 vector DNAwas digested with EcoRI, ligated into the corresponding EcoRI site inthe pACYC184 vector and transformed into E. coli DH5α cells by usingstandard methods (Ausubel et al., Short Protocols in Molecular Biology,3rd ed., Wiley 1995). E. coli were cultured as described above andkanamycin resistant transformants isolated. Plasmid DNA was isolatedfrom selected transformants and subjected to automated sequencing usingthe BigDye Terminator Cycle Sequencing Ready Reaction (Perkin Elmer) andthe ABI Prism 310 Genetic Analyzer (Perkin Elmer). Chromosomal DNAsequences at the junctions of the transposon insertions were obtained bycycle sequencing outward from the transposon using the primers5′-GGTCAGCGCAGGCGAAGCCC (BETH-F, SEQ ID NO: 1) and5′-GCCAGGTCCACACTGCCCCC (BETH-R, SEQ ID NO: 2). The results are shown inFIG. 2. An 8 by duplication was observed at the transposon-chromosomaljunction (SEQ ID NOS: 3 & 4) (FIG. 2). This duplication was alsoreported by Pelicic et al. (Proc. Natl. Acad. Sci. USA, 94:10955-10960,1997) for transpositions of this element into five different genes in M.bovis BCG. The nucleotide sequence derived from BETH-F was translatedbased on the mycobacterial codon usage. A BLAST search identified thechromosomal gene carrying the transposon insertion as homologous to theM. leprae xerC gene (GenBank No. Z97369; SEQ 113 NO: 6) encoding aputative integrase/recombinase (52% identity and 63% similarity for theregion sequenced, see FIG. 2).

Example 4 Susceptibility of Growing and Non-Growing Populations of M.paratuberculosis in Broth Culture to Antimicrobial Agents

Antimicrobial agents (antibiotics) tested were the fluoroquinolone Bay y3118, and D-cycloserine. The aminoglycoside amikacin which kills bothgrowing and non-growing M. paratuberculosis, was included as a control.M. paratuberculosis strain K-10 was grown in complete Middlebrook 7H9medium containing mycobactin J as described above to an optical density(OD₆₀₀) of 0.3 to 0.4. For non growing conditions, cells (200 ml) wereharvested at room temperature, washed in MSS (Middlebrook 7H9 saltsolution: 1.0 g/L KH₂PO₄, 0.05 g/L MgSO₄, NH₄SO₄, 2.5 g/L Na₂HPO₄,0.0005 g/L CaCl₂, 0.001 g/L ZnSO₄, and 0.001 g/L CuSO₄), and resuspendedin MSS as a single-cell suspension (Williams et al., J. Clin.Microbiol., 37:304-309, 1999). Aliquots (10 ml at a cell density of2×10⁸ CFU/mL) were placed in sterile tissue culture flasks, andantibiotics were added at the following final concentrations Bay y 3118:0.075 μg/mL (5× MIC) and 0.3 μg/mL (20× MIC); D-cyloserine: 125 μg/mL(5× MIC) and 500 μg/mL (20× MIC); amikacin: 10 μg/mL (5× MIC), and 40μg/mL (20× MC). These cultures and a control without antibiotics (0×)were incubated at 37° C. and appropriate dilutions were plated intriplicate onto complete Middlebrook 7H9 medium at various time points.When these cells were inoculated into complete Middlebrook medium(growing conditions), they displayed normal growth (growth control). Forgrowing conditions, cells were treated as described except that thegrowth control (0×) was diluted 1:10 and all cells were washed andresuspended in complete medium instead of MSS. The cells were alsoinoculated into MSS (no-growth control).

The results are shown in FIG. 3 and show that Bay y 3118 at 5× the MICdoes not kill non growing bacteria, but has a bactericidal action ongrowing bacteria. At both 5× and 20× the MIC, D-cycloserine had amoderate bactericidal effect on both growing and non growing bacteria.Amikcin, as expected, was bactericidal for both growing and non growingbacteria, but bacteria in stasis were particularly more susceptible.

Example 5 Susceptibility of Growing and Non-Growing Populations of M.paratuberculosis in Macrophage Culture to Antimicrobial Agents

Bovine macrophages of the BOMAC line (Stabel and Stabel, Vet. Immunol.Immunopathol., 45:211-220, 1995) are infected with single-cellsuspensions of mutated bacteria as described in Example 4 at a MOI of10:1. Infected BOMAC cells are then incubated at 39° C. in RPMI-1640tissue culture medium containing Bay y 3118 at 5× the MIC for threedays. After the incubation period, the infected BOMAC cells are lysedwith 0.25% SDS and the viable bacteria recovered by centrifugation.These bacteria are used to infect a fresh culture of BOMAC cells, andthis procedure is repeated until each pool of mutants has been passed 3times through BOMAC cells. The bacteria recovered from the final passageand sample from earlier passages are then subjected to molecularcharacterization as described in Example 3.

Example 6 Virulence Testing of M. paratuberculosis in Susceptible Mice

Groups of 6 to 8 week old beige mice were infected orally with nonmutated (wild type) M. paratuberculosis strain K-10 and euthanized atvarious times post infection. Bacterial loads in liver, spleen and ileumwere determined. Intestinal wall tissue was fixed and stained withhematoxylin and eosin for histological assessment. Mice were giveneither a high infectious dose of 5 doses of 1×10⁸ bacteria administeredon alternate days (Experiment I) or a low infectious dose of 1×10⁴bacteria in a single dose (Experiment II). The results are given inTable 2. With the high infectious dose, the bacteria continued to growin the spleen during the 8-week period but bacterial load in the liverleveled off at three weeks. Effective multiplication during the 8-weekperiod was also observed in the terminal ileum with an in vivogeneration time of about one week. These results showed that M.paratuberculosis can infect beige mice through the intestinal mucosa. Ahigher multiplication in the liver and spleen as compared to the ileum,however, indicated that the mice were experiencing a systemic infection.Thus, using the high infectious dose, beige mice infected with M.paratuberculosis did not parallel all the features of ruminantparatuberculosis. Under the conditions of Experiment II, the ileumdisplayed the greatest bacterial load. Bacterial multiplicationcontinued in all three organs for the 8-week period. Thus, using a lowerdose, oral infection of beige mice with M. paratuberculosis followed acourse similar to the more time consuming monoassociated nude mouseintragastric model (Hamilton et al., Vet Pathol. 28:146-155, 1991).

Example 7 Virulence Tested of Mutated Bacteria in Susceptible Mice

Beige mice are inoculated orally with a single dose of 1×10⁴ bacteria asdescribed in Example 6. For each mutant tested there is a test group ofmice that receives the mutated organism and a positive control groupthat receives the non mutated parental (wild type) strain from which themutated organisms are derived. At various times after inoculation,animals are euthanized and bacterial loads in liver, spleen and ileumare determined as described in Example 6. Mutant strains which do notshow continued multiplication in tissues as compared to thecorresponding controls are considered to be non-virulent.

Example 8 Screening of Mutant Bacteria for Ability to Stimulate Immunity

Mutant bacteria which have been found to be non virulent by the methodof Example 7 are tested to determine their ability to stimulate immunityagainst subsequent infection with non-mutated, virulent strains. Beigemice are inoculated orally with mutant non-virulent strains as describedin Example 6. A second group of mice acts as a control group andreceives only vehicle. At various times after inoculation, mice arechallenged with 1×10⁴ bacteria of the non-mutated virulent parentalstrain. At various times after challenge mice are euthanized andbacterial loads in liver, spleen and ileum are determined as describedin Example 6. Mutated strains which are found to confer immunity arefurther tested to determine optimal dosage rates and methods ofimmunization, for example, single or multiple administration oral versusparenteral administration.

TABLE 1 Ads OD₆₀₀ Ration Time Transd. Transp Kan^(r) P Expt.¹ culture²Ph/B³ (hours)⁴ Freq.⁵ Freq⁶ mutants⁷ N⁸ value⁹ I 0.48 20 4 5.4 × 10⁻⁸1.0 × 10⁻⁶ 1344 1344 26% II 0.48 200 4 5.1 × 10⁻⁹ 9.8 × 10⁻⁷ 1275 261942% III 0.38 25 6 7.0 × 10⁻⁹ 1.8 × 10⁻⁷ 176 2795 46% IV 0.38 25 24 5.4 ×10⁻⁹ 1.4 × 10⁻⁷ 136 2931 48% V 0.75 350 4 1.1 × 10⁻⁹ 3.6 × 10⁻⁵ 26895620 71% ¹Experiment number ²OD_(600 nm) of M. paratuberculosis atharvest ³Ratio of phAE94 PFU at 30° C. to the number of M.paratuberculosis K-10 CFU ⁴Adsorption time in hours at 37° C.⁵Transduction frequency: number of kanamycin-resistant colonies obtainedat 37° C. per infecting phage particle. ⁶Transposition frequency: numberof kanamycin-resistant colonies obtained at 37° C. per recipient cell.⁷Total number of kanamycin-resistance colonies obtained at 37° C.(putative transposon mutants) ⁸Cumulativce number ofkanamyucin-resistant colonies obtained ⁹Probability (%) value for therepresentation of the mutant pool assuming randon transposition.

TABLE 2 CFU/g (mean of all mice in group ± SEM) Liver Spleen Ileum WeekI II I II I II 1 5.9 ± 0.6 × 10⁷ 7.9 ± 0.4 × 10¹ 2.7 ± 0.3 × 10⁷ 1.6 ±0.3 × 10² 3.5 ± 0.6 × 10⁴ 5.9 ± 0.6 × 10³ 2 1.4 ± 0.8 × 10⁸ 1.5 ± 0.5 ×10¹ 1.1 ± 0.4 × 10⁸ 1.8 ± 0.4 × 10² 4.0 ± 0.3 × 10⁴ 2.2 ± 0.2 × 10⁴ 32.5 ± 0.4 × 10⁸ Not done 1.7 ± 0.5 × 10⁸ Not done 9.9 ± 0.6 × 10⁴ Notdone 4 Not done 3.0v0.2 × 10² Not done 1.6 ± 0.3 × 10² Not done 2.5 ±0.2 × 10⁴ 8 2.9 ± 0.6 × 10⁸ 1.1 ± 0.6 × 10³ 5.6 ± 0.4 × 10⁸ 5.9 ± 0.4 ×10³ 2.2 ± 0.3 × 10⁵ 1.3 ± 0.3 × 10⁴

CONCLUSION

In light of the detailed description of the invention and the examplespresented above, it can be appreciated that the several aspects of theinvention are achieved.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Particular formulations and processes of the presentinvention are not limited to the descriptions of the specificembodiments presented, but rather the descriptions and examples shouldbe viewed in terms of the claims that follow and their equivalents.While some of the examples and descriptions above include someconclusions about the way the invention may function, the inventors donot intend to be bound by those conclusions and functions, but put themforth only as possible explanations.

It is to be further understood that the specific embodiments of thepresent invention as set forth are not intended as being exhaustive orlimiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the are inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternativesmodifications, and variations that fall within the spirit and scope ofthe following claims.

1. A method for identifying virulence determinants of a bacteriacomprising: introducing at least one mutation into the genome of abacteria; culturing the mutated bacteria in the presence of anantimicrobial agent that kills growing but not non-growing bacteria;selecting surviving bacteria; testing the selected surviving bacteriafor virulence; selecting the non-virulent bacteria; sequencing geneticmaterial from said selected non-virulent bacteria; determining the siteof mutation; and comparing the sequence at the mutated site to thecorresponding wild type sequence.
 2. The method of claim 1 wherein saidbacteria is a mycobacteria.
 3. The method of claim 2, wherein saidmycobacteria is a slow growing mycobacteria.
 4. The method of claim 3,wherein said slow growing mycobacteria is Mycobacteriumparatuberculosis.
 5. The method of claim 1, wherein said mutation isintroduced by insertion of a transposon.
 6. The method of claim 1,wherein said mutation is a random mutation.
 7. The method of claim 1,wherein said antimicrobial agent is a fluoroquinolone.
 8. The method ofclaim 7, wherein said fluoroquinolone is Bay y
 3118. 9. The method ofclaim 8, wherein said Bay y 3118 is used at a concentration of at least0.015 μg/mL.
 10. The method of claim 1, wherein said antimicrobial isD-cycloserine.
 11. The method of claim 10, wherein said D-cycloserine isused at a concentration of at least 25.0 μg/mL.
 12. The method of claim1, wherein said mutated bacteria is cultured in an intracellular culturesystem.
 13. The method of claim 12, wherein said intracellular culturesystem is a macrophage culture system.
 14. A method for identifyingvirulence determinants in Mycobacterium paratuberculosis comprising:introducing at least one random mutation into the genome of a M.paratuberculosis bacteria by introduction of a transposon; infectingmacrophages with said mutated bacteria; culturing the macrophagescontaining said mutated bacteria in the presence of a fluoroquinolone orD-cycloserine; selecting surviving bacteria; testing the selectedsurviving bacteria for virulence in an animal; selecting thenon-virulent organisms; sequencing genetic material from said selectednon-virulent bacteria; determining the site of mutation; and comparingthe sequence at the mutated site to the corresponding wild typesequence.
 15. A method for isolating a non-virulent mutant bacteriacomprising: introducing at least one mutation into the genome of abacteria; culturing the mutated bacteria in the presence of anantimicrobial agent that kills growing but not non-growing bacteria;selecting surviving bacteria, testing the selected surviving bacteriafor virulence; and selecting the non-virulent mutant bacteria.
 16. Themethod of claim 15, wherein said bacteria is a mycobacteria
 17. Themethod of claim 15, wherein said non-virulent mutant bacteria isattenuated.
 18. The method of claim 15, wherein said antimicrobial agentis a fluoroquinolone.
 19. The method of claim 15, wherein saidfluoroquinolone is Bay y
 3118. 20. The method of claim 15, wherein saidantimicrobial is D-cycloserine.
 21. The method of claim 15, wherein saidmutation is introduced by insertion of a transposon.
 22. The method ofclaim 15, wherein said mutation is a random mutation.
 23. A compositionconsisting of a non-virulent mutant bacteria selected by the method ofclaim 15, said composition capable of stimulating an immune responseagainst a disease caused by or associated with the correspondingnon-mutated wild type.
 24. The composition of claim 23, furthercomprising a pharmaceutically acceptable carrier, diluent or excipient.25. The composition of claim 23, wherein said bacteria is amycobacteria.